Opioid peptides modulate neurotransmitter release and myocardial contraction. However, the cellular mechanisms underlying their effects are not known. We investigated the effects of micro, delta and kappa opioid receptor stimulation on the contractile properties cytosolic [Ca 2+] (Ca i) and cytosolic pH (pH i) of adult rat left ventricular myocytes. The micro agonist DAGO, (10 -5M) had no effect on the twitch. The delta agonist leucine enkephalin (10 -10M-10 -5M0 and the kappa agonist U-50,488H (10 -7- 2 x 10 -5M), had a concentration-dependent negative inotropic action. This effect was associated with a decrease in the Ca (i) transient amplitude which was monitored with the Ca 2+ probe indo-1. The sarcoplasmic reticulum (SR) Ca 2+ content was probed in the absence of electrical stimulation via the rapid addition of a high concentration of caffeine from a patch pipette above the cell. U-50,488H and leucine-enkephalin slowly increased Ca (i) or caused Ca (i) oscillations and eventually abolished the caffeine-triggered Ca (i) transient. Thus, SR Ca 2+ depletion may be a mechanism for the negative inotropic action of delta and kappa opioid receptor stimulation. Myofilament responsiveness to Ca 2+ was assessed by the relationship between contraction amplitude and systolic indo-1 transient. Leucine-enkephalin (10 -8M) had no effect while U-50,488H (10 - 5M) increased the myofilament responsiveness to Ca 2+. Thus, we examined the effects of a rapid and brief exposure to U-50,488H on cytosolic pH (pH i), which modulates myofilament sensitivity to Ca 2+. U-50,488H caused a transient increase in pH (i) measured from the change in SNARF-1 fluorescence. The pH (i) increase was abolished by: (1) blockade of the Na+/H+ exchanger by ethylisopropylamiloride and (2) inhibition of protein kinase C (PKC) activity via pretreatment with staurosporine or prolonged incubation with 4 beta-phorbol 12-myristate 13-acetate. Thus, the kappa opioid-induced increase in pHi and enhanced myofilament response to Ca 2+ in individual heart cells is likely mediated, in part at least, via PKC activation of Na+/H+ exchange.